Recombinant Escherichia coli Sensor protein ZraS (zraS)

What is the molecular structure of ZraS protein and how does it function within E. coli cells?

ZraS is a sensor histidine kinase containing 465 amino acids and located in the inner membrane of E. coli. It contains a periplasmic domain that binds zinc with high affinity, resulting in conformational changes that trigger signal transduction . ZraS functions as part of the ZraSR two-component signal transduction system (TCS), where it forms a complex with ZraP (periplasmic regulator) and ZraR (response regulator) .

The protein catalyzes several biochemical reactions critical for signal transduction:

• Autophosphorylation: ZraS [inner membrane] + ATP → ZraS-N-phospho-L-histidine [inner membrane] + ADP

• Phosphotransfer: ZraR + ZraS-N-phospho-L-histidine → ZraR-P asp56 + ZraS

These reactions enable ZraS to transduce environmental signals, particularly zinc entry into the periplasm, to cellular responses through ZraR activation.

How does the ZraSR system respond to environmental stimuli?

The ZraSR system was initially characterized as a zinc-responsive system, but recent research has revised this understanding. It's now recognized as an envelope stress response (ESR) system that uses zinc as a marker of envelope perturbation rather than directly controlling zinc homeostasis .

When envelope stress occurs, zinc enters the periplasm and binds to the periplasmic domain of ZraS, causing conformational changes that activate its histidine kinase activity . This activation leads to ZraS autophosphorylation and subsequent phosphotransfer to ZraR, initiating the cellular response to envelope stress .

Interestingly, ZraP acts as a repressor of the system when bound to zinc, creating a feedback loop that modulates system activity . Research demonstrates that despite the system's activation by zinc, the Zra proteins are not directly involved in zinc or copper resistance mechanisms .

What are the recommended methods for expressing and purifying recombinant ZraS protein?

Recombinant ZraS can be successfully expressed and purified using the following methodological approach:

• Expression system: Express full-length ZraS (1-465aa) with an N-terminal His-tag in E. coli expression systems like BL21(DE3) .

• Purification process:

  • Lyse cells in appropriate buffer conditions

  • Purify using immobilized metal affinity chromatography (IMAC)

  • Further purification may include size exclusion chromatography

• Buffer optimization: The protein is typically stored in Tris/PBS-based buffer with 6% Trehalose at pH 8.0 .

• Storage conditions: For long-term storage, add 5-50% glycerol (final concentration) and store aliquots at -20°C/-80°C. Avoid repeated freeze-thaw cycles to maintain protein integrity .

For studying just the periplasmic domain, researchers have successfully expressed and purified this domain for characterization using techniques such as multi-angle laser light scattering (MALLS), circular dichroism (CD), and differential scanning fluorescence (DSF) .

How can researchers characterize the metal-binding properties of ZraS?

The metal-binding properties of ZraS can be characterized using several complementary techniques:

When conducting these experiments, it's important to note that ZraP has been shown to bind copper with higher affinity than zinc in vitro, suggesting complex metal-binding dynamics within the ZraSR system .

How can the ZraSR system be engineered for biosensing applications?

The ZraSR system has been successfully engineered for biosensing applications, particularly for heavy metal detection. A methodological approach includes:

• Biosensor construction: A ZraP-based lead sensing and removal system can be constructed in E. coli, regulated by the ZraS/ZraR two-component system .

• Dual-function design: Researchers have developed systems that express both GFP (for sensing) and metal-binding peptides (for adsorption) under the control of the zraP promoter, enabling simultaneous detection and removal of environmental contaminants .

• Characterization methods: The expression profile of the zraP gene in response to extracellular stimuli like lead can be studied via real-time PCR to validate biosensor performance .

• Performance metrics: Engineered bacterial systems have demonstrated impressive capabilities, including:

  • Emission of fluorescence signal proportional to metal concentration

  • Adsorption capacity of up to 487 μmol lead/g cell dry cell weight (DCW)

  • Selective lead adsorption (427 μmol/g cell DCW) even in the presence of other metal ions

This approach represents a significant advance in environmental monitoring and remediation technologies based on the ZraSR system.

What role does the ZraSR system play in optimizing recombinant protein production?

The ZraSR system has emerged as a valuable tool for optimizing recombinant protein production, particularly for proteins targeted to the periplasm:

• Stress detection function: ZraS-based biosensors can detect cellular stress caused by inefficient secretion of proteins from the cytoplasm and aggregation of proteins in the periplasm .

• Production optimization: By monitoring the fluorescence fingerprint from ZraS-based biosensors, researchers can identify induction conditions that do not exceed cellular capacity and therefore do not cause cellular stress .

• Benefits for protein production:

  • More effective biomass accumulation

  • Higher titers of soluble recombinant proteins

  • Reduced formation of insoluble aggregates

• Application to disulfide-bonded proteins: This approach is particularly valuable for recombinant proteins that require disulfide bonds to fold and function correctly, as these are typically targeted to the periplasm where the necessary enzymes for disulfide bond formation exist .

By integrating ZraS-based biosensors into production processes, researchers can achieve more efficient and higher-yielding protein production systems.

How does ZraS coordinate with ZraP and ZraR to form a functional signaling system?

The ZraSR system represents a sophisticated molecular machine with three key components working in concert:

• Sensor kinase activity: Upon binding zinc, ZraS undergoes conformational changes in its periplasmic domain, triggering autophosphorylation of a conserved histidine residue in its cytoplasmic domain .

• Phosphotransfer mechanism: The phosphorylated ZraS transfers its phosphoryl group to an aspartate residue (Asp56) on ZraR, activating this response regulator to induce expression of target genes .

• Regulatory feedback loop: ZraP functions as a periplasmic repressor partner. When bound to zinc, ZraP represses the expression of the zraPSR operon, creating a negative feedback loop that modulates system activity .

• Metal binding specificity: Interestingly, while ZraS responds to zinc in vivo, in vitro studies have shown that ZraP binds copper with a higher affinity than zinc, suggesting complex metal-binding dynamics within the system .

• Chaperone activity: ZraP also displays chaperone properties that are partially dependent on zinc binding, contributing to the cell's ability to respond to envelope stress .

This integrated mechanism allows zinc to function as a marker of envelope stress perturbation, with the ZraPSR system acting as a sentinel that senses and responds to zinc entry into the periplasm .

What are the structural features of ZraS that enable its sensory function?

ZraS contains several structural domains that contribute to its sensory function:

• Periplasmic domain: This domain contains the zinc-binding site responsible for detecting environmental signals. Upon binding zinc, this domain undergoes conformational changes that propagate to the cytoplasmic domains .

• Transmembrane regions: ZraS contains transmembrane helices that anchor it in the inner membrane and transmit signals from the periplasm to the cytoplasm .

• Histidine kinase domain: Located in the cytoplasmic portion, this domain contains the conserved histidine residue that becomes phosphorylated during signal transduction .

• ATP-binding domain: This region binds ATP, providing the phosphoryl group for the autophosphorylation reaction .

• Metal-binding sites: Studies have shown that ZraS binds one zinc molecule with high affinity, resulting in conformational changes consistent with a triggering function of the metal ion .

The complete amino acid sequence of ZraS (465 amino acids) includes regions responsible for these various functions:
MRFMQRSKDSLAKWLSAILPVVIVGLVGLFAVTVIRDYGRASEADRQALLEKGNVLIRAL ESGSRVGMGMRMHHVQQQALLEEMAGQPGVLWFAVTDAQGIIILHSDPDKVGRALYSPDE MQKLKPEENSRWRLLGKTETTPALEVYRLFQPMSAPWRHGMHNMPRCNGKAVPQVDAQQA IFIAVDASDLVATQSGEKRNTLIILFALATVLLASVLSFFWYRRYLRSRQLLQDEMKRKE KLVALGHLAAGVAHEIRNPLSSIKGLAKYFAERAPAGGEAHQLAQVMAKEADRLNRVVSE LLELVKPTHLALQAVDLNTLINHSLQLVSQDANSREIQLRFTANDTLPEIQADPDRLTQV LLNLYLNAIQAIGQHGVISVTASESGAGVKISVTDSGKGIAADQLDAIFTPYFTTKAEGT GLGLAVVHNIVEQHGGTIQVASQEGKGSTFTLWLPVNITRKDPQG

How does ZraS compare with other two-component systems involved in envelope stress response?

ZraS operates within a broader network of envelope stress response systems in E. coli:

• Functional homology with CpxPAR: The ZraPSR system is functionally homologous to the CpxPAR system, which integrates signals of envelope perturbation, including misfolded periplasmic proteins .

• Mechanistic differences: While both systems respond to envelope stress, they differ in their activation mechanisms:

  • CpxA directly senses misfolded proteins

  • ZraS uses zinc as an indirect marker of envelope stress

• Regulatory patterns: The auxiliary periplasmic regulator CpxP inhibits the Cpx pathway by interacting with CpxA. Similarly, ZraP inhibits the Zra pathway, though through different molecular mechanisms .

• Stress specificity: Different two-component systems respond to distinct types of envelope stress:

  • ZraSR: Responds to zinc entry into the periplasm

  • CpxAR: Responds to misfolded proteins

  • BaeSR: Responds to envelope-damaging compounds

  • RcsCDB: Responds to outer membrane and peptidoglycan stress

• Integrated response: These systems likely form an integrated network that enables the cell to mount appropriate responses to various types of envelope stress .

What challenges remain in understanding the full mechanism of ZraS activation and signaling?

Despite significant advances, several challenges remain in fully understanding ZraS functionality:

• Structural characterization: While the periplasmic domain has been studied, the full-length structure of ZraS, particularly in different activation states, remains to be determined .

• Signal transmission mechanism: How zinc binding in the periplasmic domain triggers conformational changes that activate the cytoplasmic kinase domain is not fully understood at the molecular level .

• Metal-binding specificity: The observation that ZraP binds copper with higher affinity than zinc in vitro raises questions about the true metal specificity of the system and potential roles in sensing multiple metals .

• Physiological triggers: Beyond zinc, the full range of physiological conditions that activate the ZraSR system in vivo requires further investigation .

• Integration with other stress responses: How the ZraSR system coordinates with other envelope stress response systems to mount an appropriate cellular response remains to be fully elucidated .

• Target genes: The complete set of genes regulated by activated ZraR and their roles in responding to envelope stress need further characterization .

Addressing these challenges will require interdisciplinary approaches combining structural biology, biochemistry, genetics, and systems biology to fully elucidate the molecular mechanisms and physiological roles of the ZraSR system.